The in vitro perfused rabbit ovary has served as a useful model for the study of ovulation. It allows removal of the ovary from systemic influences, inclusion in the perfusate of agents to be evaluated for their influence on ovulation, collection of aliquots of perfusion medium to determine biochemical changes associated with follicular growth and rupture, direct observation of ovulation, and collection of the ova from ruptured and unruptured follicles to determine the degree of maturation. In the current year, this system will be used to further study the role of calcium in the ovulatory process using calcium-free medium and replacement with calcium. The effect of histamine on follicle rupture and ovum maturation will also be examined in the in vitro system. Histamine content of medium aliquots will be determined at various time intervals in these experiments. To further expand on the role of protaglandins in ovulation, localization of the site of PG production within ovarian tissue corresponding to time of ovulation will be attempted using immunofluorescent antibody technique. In addition, PGE2 will be added to the perfusion fluid to determine its action on follicle rupture and ovum maturation. Validation of the perfused rabbit ovary system for further studies includes successful fertilization, both in vivo and in vitro of ovulated oocytes and production of offspring. Fertilizability of ova recovered from this system will be evaluated, thus indicating that follicle rupture and ovum development are occurring in a viable system. These related studies will provide additional information involving local ovarian factors influencing ovulatory phenomena.